Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis

Abstract

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

FIG. 1

Primers for amplification of fungal 18S-rDNA sequences. Primer pair EF4-EF3 (fragment PCR1a) is specific for fungal 18S rDNA, and primer pair EF4-fung5 (fragment PCR1b) amplifies a smaller, 550-bp fragment specific for fungi. Primer pair EF4-NS3 (PCR2) can be used directly on clones or in a nested approach on PCR1 products for community analysis by TGGE. The primers which were developed in this work are given in alignment with 18S-rDNA sequences of species from all major fungal taxa.

FIG. 2

TGGE of amplified 18S-rDNA fragments representing the fungal community in bulk and rhizophere soil samples of the microcosm experiment. Banding patterns were obtained by mixing the duplicate samples before PCR and adding PCR products from both primer pairs (EF4-EF3 and EF4-fung5) the same lane. Lane 1, day 5 bulk soil; lane 2, day 5 rhizosphere soil; lane 3, day 10 bulk soil; and lane 4, day 10 rhizosphere soil. Numbered bands are explained in the text.

FIG. 3

A dendrogram based on the presence or absence of bands (Fig. 2) was constructed to represent the percentages of genetic identity between the profiles of the microcosm samples of bulk and rhizosphere (rhizo) soils.